Microglia microvesicles contained microRNA-based methods for the diagnosis, prognosis and treatment monitoring of neurological, neurodegenerative and inflammation-based diseases

ABSTRACT

The present invention describes a method for the in vitro diagnosis, prognosis and/or treatment monitoring of neurodegenerative, neurological and inflammation-based diseases, wherein the method comprises the steps: a) isolating microglial microvesicles (MVs) from biological fluids obtained from an individual; b) collecting the microRNA (miRNA) contained into said MVs; c) determining the expression profile of a predetermined set of miRNA; d) comparing said expression profile to one or several reference expression profiles, wherein the comparison of said determined expression profile to said one or several reference expression profiles allows for the diagnosis, prognosis and/or treatment monitoring of the disease.

This is a national stage application filed under 35 U.S.C. § 371 ofinternational application PCT/EP2016/078190, filed under the authorityof the Patent Cooperation Treaty on Nov. 18, 2016, published; whichclaims the benefit of Italy Patent Application No. 102015000074820,filed on Nov. 20, 2015. The entire disclosures of all the aforementionedapplications are expressly incorporated herein by reference for allpurposes.

The present invention describes a method for the in vitro diagnosis,prognosis and/or treatment monitoring of neurodegenerative, neurologicaland inflammation-based diseases, wherein the method comprises the steps:

-   -   a) isolating microglial microvesicles (MVs) from biological        fluids obtained from an individual;    -   b) collecting the microRNA (miRNA) contained into said MVs;    -   c) determining the expression profile of a predetermined set of        miRNA;    -   d) comparing said expression profile to one or several reference        expression profiles,        wherein the comparison of said determined expression profile to        said one or several reference expression profiles allows for the        diagnosis, prognosis and/or treatment monitoring of the disease.

BACKGROUND

Emerging evidence indicates that inflammation represents a pathogenicfactor in many CNS diseases, including chronic neurodegenerativediseases such as Alzheimer's Disease (AD), Parkinson's Disease (PD),Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS),neurological disorders such as schizophrenia or epilepsy but also rarediseases such as Batten's Disease. Evidence indicating a role forinflammation in the early phases of brain tumors development have beenreported, too (Sowers J L et al. The role of inflammation in braincancer. Adv Exp Med Biol. 2014; 816:75-105).

Microglia, the resident immune cells in the brain, plays a crucial rolein the onset of neuroinflammation. Microglial cells are the majorcellular elements with immune function inside the CNS and arefundamental in orchestrating inflammatory brain responses to externalchallenges.

In spite of the evidence indicating that chronic inflammation mightinfluence the pathogenesis of degenerative diseases, the mechanisms ofcommunication between microglia and neurons have not been clearlyelucidated, and it is still unclear which molecules are being releasedby these cells and how the damage occurs at neuronal level. Activatedmicroglia may affect either positively or negatively neuronal survival,via the production of growth factors or pro-inflammatory mediators.

Upon cellular activation, microglia release plasma membrane-derivedmicrovesicles (MVs) at a very early stage of the inflammatory processleading to neurodegeneration (Bianco F et al. Astrocyte-derived ATPinduces vesicle shedding and IL-1 beta release from microglia. JImmunol. 2005 174(11):7268-77; Bianco F et al. Acid sphingomyelinaseactivity triggers microparticle release from glial cells. EMBO J. 200928(8):1043-54).

MVs have been found in high number in the cerebrospinal fluid ofpatients with mild cognitive impairment (Agosta F et al. Myeloidmicrovesicles in cerebrospinal fluid are associated with myelin damageand neuronal loss in mild cognitive impairment and Alzheimer disease.Ann Neurol. 2014 76(6):813-25; U.S. Pat. No. 8,999,655 B2).

MicroRNAs (miRNAs, miR) are small non-coding RNAs expressed in animalsand plants. They regulate cellular function, cell survival, cellactivation and cell differentiation during development. Many miRNAs areconserved in sequence between distantly related organisms.

A diagnostic method for neurodegenerative, neurological andinflammation-based diseases is strongly needed, as well as a method formonitoring the therapeutic outcome in diseases where the therapeuticprotocol has to be strictly defined and punctually modified accordinglyto the specific reaction observed in each treated individual.

DESCRIPTION OF THE INVENTION

The authors of the present invention have surprisingly demonstrated astrong and specific correlation between miRNA content and profile of MVsderived from microglia cells and neurodegenerative, neurological andinflammation-based diseases. The authors have surprisingly demonstratedthat specific patterns of miRNAs are activated in microglial MVs derivedunder different detrimental conditions.

It is here firstly described a pathology and/or disease-specific miRNAprofile within the microglial MVs isolated from biological fluids bothfor early diagnosis, prognosis and/or treatment monitoring.

A “miRNA” is a naturally occurring, small non-coding RNA that is about17 to about 25 nucleotide (nt) in length in its biologically active formthat negatively regulates mRNA translation on a sequence-specificmanner. Identified miRNAs are registered in the miRNA database miRBase(http://microma.sanger.ac.uk/).

A “sample”, as defined herein, is a small part of a subject,representative of the whole and may be constituted by a body fluidsample. Body fluid samples may be blood, plasma, serum, urine, sputum,cerebrospinal fluid, milk, or ductal fluid samples and may likewise befresh, frozen or fixed. Samples may be removed surgically, by extractioni.e. by hypodermic or other types of needles, by microdissection orlaser capture. The sample should contain any biological materialsuitable for detecting the desired biomarker (miRNA), thus, said sampleshould advantageously comprise cell material from the subject.

A “reference sample”, as used herein, means a sample obtained fromindividuals, preferably two or more individuals, known to be free of anyneurodegenerative, neurological disease or neuroinflammation or from thegeneral population. The suitable reference expression levels of miRNAscan be determined by measuring the expression levels of said miRNAs inseveral suitable individuals, and such reference levels can be adjustedto specific populations. In a preferred embodiment, the reference sampleis obtained from a pool of healthy individuals. The expression profileof the miRNAs in the reference sample can, preferably, be generated froma population of two or more individuals; for example, the population cancomprise 3, 4, 5, 10, 15, 20, 30, 40, 50 or more subjects.

An “individual”, as used herein, refers to a mammal, human or non-human,under observation, preferably a human being. The individual may be anyindividual, an individual predisposed to a neuro-related disease or anindividual suffering from a neuro-related disease.

As used herein, the expression “diagnosis” or “diagnosing” relates tomethods by which the skilled person can estimate and even determinewhether or not an individual is suffering from a given disease orcondition.

Along with diagnosis, clinical disease prognosis is also an area ofgreat concern and interest. It is important to know the stage andrapidity of advancement of the disease in order to plan the mosteffective therapy. If a more accurate prognosis can be made, appropriatetherapy, and in some instances less severe therapy for the patient canbe chosen.

Further, the expression “method of diagnosing” as used herein relates toa method that may essentially consist of the steps mentioned below, ormay include additional steps. However, it must be understood that themethod, in a preferred embodiment, is a method that is carried out invitro, i.e., it is not carried out in the human or animal body.

It has here surprisingly found that the miRNAs contained in microglialMVs, microglial MVs that can be isolated according to procedures knownin the state of the art, reflect in a reproducible manner any variationfrom the physiological state in the CNS. CNS impairments due toneurodegenerative diseases, but also linked to neurological disorders orneuroinflammation are each linked to a very specific and reproduciblemiRNA profile. The variation from the physiological miRNAs profile ofmicroglial MVs miRNAs is very sensitive to disease progression,therefore making said miRNA a very useful tool not only for an earlydiagnosis but also for therapeutic monitoring.

The here proposed methodology offers the great advantage to makepossible a miRNA based analysis where miRNA are obtained from a singlecellular subpopulation, i.e. from microglial MVs. This is extremelyadvantageous because the process to isolate microglial MVs allows toisolate only microglial MVs, while other methodologies currentlyavailable lead to the obtainment of vesicles from several heterogeneouscellular populations. Therefore, when data are obtained from microglialMVs their reproducibility, sensitivity and specificity is increased.

In a first embodiment, the present invention describes a method for thein vitro diagnosis or clinical disease prognosis of a neurodegenerative,neurological or inflammation-based disease, wherein the method comprisesthe steps:

-   -   a) isolating microglial MVs from biological fluids obtained from        an individual;    -   b) collecting the miRNA contained into said MVs;    -   c) determining the expression profile of a predetermined set of        miRNA;    -   d) comparing said expression profile to one or several reference        sample expression profiles,        wherein the comparison of said determined expression profiles to        said one or several reference sample expression profiles allows        for the diagnosis or prognosis of the disease.        In a preferred embodiment, the disease is selected form the        group comprising: AD, PD, ALS, MS, Batten's Disease,        Schizophrenia, Epilepsy, Neuropathic pain, Neuroinflammation,        Tourette Syndrome, HD, Autism, Rett Syndrome, Depression,        Ischemia, Glioblastoma, Meningitis, Traumatic Brain Injury.        Glioblastoma are included because there are evidences suggesting        that subpopulations of cells within human gliomas, specifically        GBM (glioblastoma multiforme), are neoplastic        macrophages/microglia (Leanne C Huysentruyt et al. Hypothesis:        are neoplastic macrophages/microglia present in glioblastoma        multiforme? ASN Neuro. 2011; 3(4)).

In a preferred embodiment, said expression profile is determined ofmiRNAs selected from the group consisting of miR-125a-5p (SEQ ID 1),miR-300-3p (SEQ ID 2), miR-330-3p (SEQ ID 3), miR-466n-3p (SEQ ID 4),miR-501-5p (SEQ ID 5), miR-146a-5p (SEQ ID 6), miR-24-1-5p (SEQ ID 7),miR-1306-5p (SEQ ID 8), miR-744-5p (SEQ ID 9), miR-671-5p (SEQ ID 10),miR-134-5p (SEQ ID 11), miR-877-5p (SEQ ID 12), miR-23b-5p (SEQ ID 13),miR-669c-5p (SEQ ID 14), miR-29b-3p (SEQ ID 15), miR-195a-5p (SEQ ID16), miR-151-5p (SEQ ID 17), miR-374c-3p (SEQ ID 18), miR-6539 (SEQ ID19), miR-16-1-3p (SEQ ID 20), miR-6399 (SEQ ID 21), miR-6240 (SEQ ID22), miR-23a-5p (SEQ ID 23), miR-92a-1-5p (SEQ ID 24), miR-219a-1-3p(SEQ ID 25), miR-128-1-5p (SEQ ID 26), miR-1949 (SEQ ID 27), miR-872-3p(SEQ ID 28), miR-582-3p (SEQ ID 29), miR-338-5p (SEQ ID 30), miR-379-5p(SEQ ID 31), miR-155-5p (SEQ ID 32), miR-450a-5p (SEQ ID 33), miR-100-5p(SEQ ID 34), miR-152-3p (SEQ ID 35), miR-222-3p (SEQ ID 36), let-7e (SEQID 37), miR-18b (SEQ ID 38), miR-19a (SEQ ID 39), miR-21b (SEQ ID 40),miR-26b (SEQ ID 41), miR-29b-1 (SEQ ID 42), miR-30c-1 (SEQ ID 43),miR-100 (SEQ ID 44), miR-130a (SEQ ID 45), miR-181c (SEQ ID 46),miR-297a-1 (SEQ ID 47), miR-330 (SEQ ID 48), miR-342 (SEQ ID 49),miR-484 (SEQ ID 50), miR-669b (SEQ ID 51), miR-669e (SEQ ID 52), miR-708(SEQ ID 53), miR-146b (SEQ ID 54), miR-188 (SEQ ID 55), miR-346 (SEQ ID56), miR-466f-3 (SEQ ID 57), miR-541 (SEQ ID 58), miR-706 (SEQ ID 59),miR-712 (SEQ ID 60), miR-714 (SEQ ID 61), miR-1224 (SEQ ID 62), miR-10b(SEQ ID 63), mir-22 (SEQ ID 64), mir-23b (SEQ ID 65), mir-132 (SEQ ID66), mir-148b (SEQ ID 67), mir-154 (SEQ ID 68), mir-183 (SEQ ID 69),mir-337 (SEQ ID 70), mir-19b-1 (SEQ ID 71), mir-30a (SEQ ID 72), mir-33(SEQ ID 73), mir-99b (SEQ ID 74), mir-144 (SEQ ID 75), mir-151 (SEQ ID76), mir-182 (SEQ ID 77), mir-223 (SEQ ID 78), mir-340 (SEQ ID 79),mir-374b (SEQ ID 80), mir-432 (SEQ ID 81), mir-1247 (SEQ ID 82),let-7a-2 (SEQ ID 83), mir-30b (SEQ ID 84), mir-103-2 (SEQ ID 85),mir-107 (SEQ ID 86), mir-142a (SEQ ID 87), mir-146a (SEQ ID 88),mir-374c (SEQ ID 89), mir-126b (SEQ ID 90), mir-134 (SEQ ID 91), mir-320(SEQ ID 92), let-7a-1 (SEQ ID 93), mir-34a (SEQ ID 94), mir-92b (SEQ ID95), mir-211 (SEQ ID 96), let-7f-1 (SEQ ID 97), mir-19b-2 (SEQ ID 98),mir-137 (SEQ ID 99), mir-155 (SEQ ID 100), mir-219b (SEQ ID 101),mir-338 (SEQ ID 102), mir-376c (SEQ ID 103), mir-379 (SEQ ID 104),mir-451a (SEQ ID 105), mir-494 (SEQ ID 106), mir-17 (SEQ ID 107),mir-20a (SEQ ID 108), let-7c-1 (SEQ ID 109), mir-20b (SEQ ID 110),mir-145a (SEQ ID 111), mir-186 (SEQ ID 112), mir-664 (SEQ ID 113),mir-122 (SEQ ID 114), mir-409 (SEQ ID 115), miR-199b (SEQ ID 116),mir-221 (SEQ ID 117), mir-296 (SEQ ID 118), mir-329 (SEQ ID 119),mir-382 (SEQ ID 120), mir-29c (SEQ ID 121), mir-128-1 (SEQ ID 122),mir-138-1 (SEQ ID 123), mir-218-1 (SEQ ID 124), mir-222 (SEQ ID 125),mir-344-1 (SEQ ID 126), mir-466b-2 (SEQ ID 127), mir-674 (SEQ ID 128),mir-207 (SEQ ID 129), mir-18a (SEQ ID 130), mir-448 (SEQ ID 131),mir-146b-3 (SEQ ID 132), mir-669c (SEQ ID 133), let-7d (SEQ ID 134),mir-30e (SEQ ID 135), mir-34b (SEQ ID 136), mir-(SEQ ID 137), mir-124-1(SEQ ID 138), mir-181a-1 (SEQ ID 139), mir-181b-1 (SEQ ID 140), mir-181d(SEQ ID 141), mir-185 (SEQ ID 142), mir-187 (SEQ ID 143), mir-190a (SEQID 144), mir-191 (SEQ ID 145), mir-301a (SEQ ID 146), mir-325 (SEQ ID147), mir-331 (SEQ ID 148), mir-345 (SEQ ID 149), mir-361 (SEQ ID 150),mir-380 (SEQ ID 151), mir-381 (SEQ ID 152), mir-450a-2 (SEQ ID 153),mir-497a (SEQ ID 246), mir-497b (SEQ ID 155), mir-505 (SEQ ID 156),mir-551b (SEQ ID 157), mir-742 (SEQ ID 158), mir-875 (SEQ ID 159),mir-935 (SEQ ID 160), mir-21a (SEQ ID 161), mir-24-2 (SEQ ID 162),mir-27b (SEQ ID 163), mir-31 (SEQ ID 164), mir-34c (SEQ ID 165),mir-129-1 (SEQ ID 166), mir-140 (SEQ ID 167), mir-142b (SEQ ID 168),mir-148a (SEQ ID 169), mir-152 (SEQ ID 170), mir-184 (SEQ ID 171),mir-199a-1 (SEQ ID 172), mir-204 (SEQ ID 173), mir-212 (SEQ ID 174),mir-214 (SEQ ID 175), mir-375 (SEQ ID 176), mir-455 (SEQ ID 177),mir-711 (SEQ ID 178), mir-882 (SEQ ID 179), mir-192 (SEQ ID 180),mir-219a-1 (SEQ ID 181), mir-383 (SEQ ID 182), mir-542 (SEQ ID 183),mir-700 (SEQ ID 184), mir-705 (SEQ ID 185), mir-762 (SEQ ID 186),mir-1901 (SEQ ID 187), mir-1928 (SEQ ID 188), mir-3474 (SEQ ID 189),mir-105 (SEQ ID 190), mir-141 (SEQ ID 191), mir-200c (SEQ ID 192),mir-201 (SEQ ID 193), mir-297b (SEQ ID 194), mir-302c (SEQ ID 195),mir-495 (SEQ ID 196), mir-670 (SEQ ID 197), mir-673 (SEQ ID 198),mir-1934 (SEQ ID 199), mir-129b (SEQ ID 200), mir-328 (SEQ ID 201),mir-487b (SEQ ID 202), let-7b (SEQ ID 203), mir-292b (SEQ ID 204),mir-125b-2 (SEQ ID 205), mir-365-1 (SEQ ID 206), mir-32 (SEQ ID 207),mir-125a (SEQ ID 208), mir-128-2 (SEQ ID 209), mir-135a-1 (SEQ ID 210),mir-139 (SEQ ID 211), mir-149 (SEQ ID 212), mir-181a-2 (SEQ ID 213),mir-326 (SEQ ID 214), mir-483 (SEQ ID 215), mir-491 (SEQ ID 216), let-7c(SEQ ID 217), mir-9-1 (SEQ ID 218), mir-15b (SEQ ID 219), mir-16-1 (SEQID 220), mir-21 (SEQ ID 221), mir-23a (SEQ ID 222), mir-24-1 (SEQ ID223), mir-25 (SEQ ID 224), mir-27a (SEQ ID 225), mir-92a-1 (SEQ ID 226),mir-93 (SEQ ID 227), mir-103a-1 (SEQ ID 228), mir-106b (SEQ ID 229),mir-125b-1 (SEQ ID 230), mir-150 (SEQ ID 231), mir-210 (SEQ ID 232),mir-718 (SEQ ID 233), mir-335 (SEQ ID 234), mir-297 (SEQ ID 235),mir-466 (SEQ ID 236), mir-151a (SEQ ID 237), mir-126 (SEQ ID 238),mir-320a (SEQ ID 239), mir-329-5p (SEQ ID 240), mir-497-5p (SEQ ID 241),mir-664a-5p (SEQ ID 242), mir-21-5p (SEQ ID 243), mir-142-5p (SEQ ID244), mir-129-5p (SEQ ID 245), mir-146b (SEQ ID 154).

In a further preferred embodiment, said method is a method for diagnosisor prognosis of PD in an individual, wherein a pattern of at least 11up-regulated and least 7 down-regulated specific miRNAs listed above isan indicator of PD, preferably said pattern is the pattern listed inTable 1.

TABLE 1 PD, microglial MVs miRNA pattern (+, upregulated; −,downregulated) miRNA miR-100-5p (SEQ ID 34) − miR-128-1-5p (SEQ ID 26) +miR-152-3p (SEQ ID 35) − miR-155-5p (SEQ ID 32) − miR-16-1-3p (SEQ ID20) + miR-1949 (SEQ ID 27) − miR-219a-1-3p (SEQ ID 25) + miR-222-3p (SEQID 36) + miR-23a-5p (SEQ ID 23) + miR-338-5p (SEQ ID 30) + miR-379-5p(SEQ ID 31) + miR-450a-5p (SEQ ID 33) + miR-501-5p (SEQ ID 5) −miR-582-3p (SEQ ID 29) + miR-6240 (SEQ ID 22) + miR-6399 (SEQ ID 21) −miR-872-3p (SEQ ID 28) − miR-92a-1-5p (SEQ ID 24) +In a preferred embodiment, said pattern is the pattern listed in Table1a.

TABLE 1a PD, microglial MVs miRNA pattern (+, upregulated; −,downregulated) miRNA miR-100-5p (SEQ ID 34) − miR-128-1-5p (SEQ ID 26) +miR-152-3p (SEQ ID 35) − miR-155-5p (SEQ ID 32) − miR-16-1-3p (SEQ ID20) + miR-219a-1-3p (SEQ ID 25) + miR-222-3p (SEQ ID 36) + miR-23a-5p(SEQ ID 23) + miR-338-5p (SEQ ID 30) + miR-379-5p (SEQ ID 31) +miR-450a-5p (SEQ ID 33) + miR-501-5p (SEQ ID 5) − miR-582-3p (SEQ ID29) + miR-92a-1-5p (SEQ ID 24) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of AD in an individual, wherein a pattern of at least 12up-regulated and at least 4 down-regulated specific miRNAs listed aboveis an indicator of AD, preferably said pattern is the pattern listed inTable 2.

TABLE 2 AD, microglial MVs pattern (+, upregulated; −, downregulated)miRNA miR-501-5p (SEQ ID 5) + miR-125a-5p (SEQ ID 1) + miR-1306-5p (SEQID 8) + miR-134-5p (SEQ ID 11) + miR-146a-5p (SEQ ID 6) + miR-151-5p(SEQ ID 17) − miR-23b-5p (SEQ ID 13) − miR-24-1-5p (SEQ ID 7) +miR-300-3p (SEQ ID 2) + miR-330-3p (SEQ ID 3) + miR-374c-3p (SEQ ID 18)− miR-466n-3p (SEQ ID 4) + miR-669c-5p (SEQ ID 14) − miR-671-5p (SEQ ID10) + miR-744-5p (SEQ ID 9) + miR-877-5p (SEQ ID 12) +In a preferred embodiment, said pattern is the pattern listed in Table2a.

TABLE 2a AD, microglial MVs pattern (+, upregulated; −, downregulated)miRNA miR-501-5p (SEQ ID 5) + miR-125a-5p (SEQ ID 1) + miR-1306-5p (SEQID 8) + miR-134-5p (SEQ ID 11) + miR-146a-5p (SEQ ID 6) + miR-151-5p(SEQ ID 17) − miR-23b-5p (SEQ ID 13) − miR-24-1-5p (SEQ ID 7) +miR-300-3p (SEQ ID 2) + miR-330-3p (SEQ ID 3) + miR-374c-3p (SEQ ID 18)− miR-671-5p (SEQ ID 10) + miR-744-5p (SEQ ID 9) + miR-877-5p (SEQ ID12) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of ischemia in an individual, wherein a pattern of at least16, more preferably at least 20 of the miRNA listed in Table 3 is anindicator of ischemia, still more preferably said pattern is the patternlisted in Table 3.

TABLE 3 ischemia, microglial MVs pattern (+, upregulated; −,downregulated) miRNA let-7e (SEQ ID 37) − mir-18b (SEQ ID 38) − mir-19a(SEQ ID 39) − mir-21 (SEQ ID 221) − mir-26b (SEQ ID 41) − mir-29b-1 (SEQID 42) − mir-30c-1 (SEQ ID 43) − mir-100 (SEQ ID 44) − mir-130a (SEQ ID45) − mir-181c (SEQ ID 46) − mir-297 (SEQ ID 235) − mir-330 (SEQ ID 48)− mir-342 (SEQ ID 49) − mir-484 (SEQ ID 50) − mir-669b (SEQ ID 51) −mir-669e (SEQ ID 52) − mir-708 (SEQ ID 53) − mir-146b (SEQ ID 54) +mir-188 (SEQ ID 55) + mir-346 (SEQ ID 56) + mir-466 (SEQ ID 236) +mir-541 (SEQ ID 58) + mir-706 (SEQ ID 59) + mir-712 (SEQ ID 60) +mir-714 (SEQ ID 61) + mir-1224 (SEQ ID 62) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of Tourette's Syndrome in an individual, wherein a patternof at least 13, preferably at least 16 of the miRNA listed in Table 4 isan indicator of Tourette's Syndrome, preferably said pattern is thepattern listed in Table 4.

TABLE 4 Tourette's Syndrome, microglial MVs pattern (+, upregulated; −,downregulated) miRNA mir-10b (SEQ ID 63) − mir-22 (SEQ ID 64) − mir-23b(SEQ ID 65) − mir-132 (SEQ ID 66) − mir-148b (SEQ ID 67) − mir-154 (SEQID 68) − mir-183 (SEQ ID 69) − mir-337 (SEQ ID 70) − mir-1224 (SEQ ID62) − mir-19b-1 (SEQ ID 71) + mir-30a (SEQ ID 72) + mir-33 (SEQ ID 73) +mir-99b (SEQ ID 74) + mir-144 (SEQ ID 75) + mir-151a (SEQ ID 237) +mir-182 (SEQ ID 77) + mir-223 (SEQ ID 78) + mir-340 (SEQ ID 79) +mir-374b (SEQ ID 80) + mir-432 (SEQ ID 81) + mir-1247 (SEQ ID 82) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of neuropathic pain in an individual, wherein a pattern ofat least 8, preferably at least 10 of the miRNA listed in Table 5 is anindicator of neuropathic pain, preferably said pattern is the patternlisted in Table 5.

TABLE 5 Neurophatic pain, microglial MVs pattern (+, upregulated; −,downregulated) miRNA let-7a-2 (SEQ ID 83) − mir-30b (SEQ ID 84) −mir-103-2 (SEQ ID 85) − mir-107 (SEQ ID 86) − mir-142a (SEQ ID 87) −mir-146a (SEQ ID 88) − mir-151 (SEQ ID 76) − mir-374c (SEQ ID 89) −mir-126 (SEQ ID 238) + mir-134 (SEQ ID 91) + mir-320a (SEQ ID 239) +mir-374b (SEQ ID 80) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of autism in an individual, wherein a pattern of at least13, preferably at least 16 of the miRNA listed in Table 6 is anindicator of autism, preferably said pattern is the pattern listed inTable 6.

TABLE 6 Autism, microglial MVs pattern (+, upregulated; −,downregulated) miRNA let-7a-1 (SEQ ID 93) − mir-34a (SEQ ID 94) −mir-92b (SEQ ID 95) − mir-211 (SEQ ID 96) − let-7f-1 (SEQ ID 97) +mir-19a (SEQ ID 39) + mir-19b-2 (SEQ ID 98) + mir-21 (SEQ ID 221) +mir-22 (SEQ ID 64) + mir-137 (SEQ ID 99) + mir-142a (SEQ ID 87) +mir-144 (SEQ ID 75) + mir-146b (SEQ ID 154) + mir-155 (SEQ ID 100) +mir-219b (SEQ ID 101) + mir-338 (SEQ ID 102) + mir-376c (SEQ ID 103) +mir-379 (SEQ ID 104) + mir-451a (SEQ ID 105) + mir-494 (SEQ ID 106) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of multiple sclerosis in an individual, wherein a patternof at least 7 of the miRNA listed in Table 7 is an indicator of multiplesclerosis, preferably said pattern is the pattern listed in Table 7.

TABLE 7 multiple sclerosis, microglial MVs pattern (+, upregulated; −,downregulated) miRNA mir-17 (SEQ ID 107) − mir-20a (SEQ ID 108) −let-7c-1 (SEQ ID 109) + mir-20b (SEQ ID 110) + mir-142a (SEQ ID 87) +mir-145a (SEQ ID 111) + mir-186 (SEQ ID 112) + mir-223 (SEQ ID 78) +mir-664 (SEQ ID 113) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of Rett Syndrome in an individual, wherein a pattern of atleast 10 of the miRNA listed in Table 8 is an indicator of RettSyndrome, preferably said pattern is the pattern listed in Table 8.

TABLE 8 Rett Syndrome, microglial MVs pattern (+, upregulated; −,downregulated) miRNA mir-122 (SEQ ID 114) − mir-130a (SEQ ID 45) −mir-146a (SEQ ID 88) − mir-146b (SEQ ID 54) − mir-342 (SEQ ID 49) −mir-409 (SEQ ID 115) − mir-29b-1 (SEQ ID 42) + mir-92b (SEQ ID 95) +mir-199b (SEQ ID 116) + mir-221 (SEQ ID 117) + mir-296 (SEQ ID 118) +mir-329-5p (SEQ ID 240) + mir-382 (SEQ ID 120) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of Huntington's Disease in an individual, wherein a patternof at least 12 of the miRNA listed in Table 9 is an indicator ofHuntington's Disease, preferably said pattern is the pattern listed inTable 9.

TABLE 9 Huntington's Disease, microglial MVs pattern (+, upregulated; −,downregulated) miRNA mir-22 (SEQ ID 64) − mir-29c (SEQ ID 121) −mir-128-1 (SEQ ID 122) − mir-132 (SEQ ID 66) − mir-138-1 (SEQ ID 123) −mir-218-1 (SEQ ID 124) − mir-222 (SEQ ID 125) − mir-344-1 (SEQ ID 126) −mir-466 (SEQ ID 236) − mir-674 (SEQ ID 128) − mir-34a (SEQ ID 94) +mir-207 (SEQ ID 129) + mir-18a (SEQ ID 130) + mir-448 (SEQ ID 131) +mir-669c (SEQ ID 133) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of epilepsy in an individual, wherein a pattern of at least31, preferably at least 42 of the miRNA listed in Table 10 is anindicator of epilepsy, preferably said pattern is the pattern listed inTable 10.

TABLE 10 epilepsy, microglial MVs pattern (+, upregulated; −,downregulated) miRNA let-7d (SEQ ID 134) − let-7f-1 (SEQ ID 97) −mir-30a (SEQ ID 72) − mir-30e (SEQ ID 135) − mir-34b (SEQ ID 136) −mir-98 (SEQ ID 137) − mir-124-1 (SEQ ID 138) − mir-181a-1 (SEQ ID 139) −mir-181b-1 (SEQ ID 140) − mir-181d (SEQ ID 141) − mir-185 (SEQ ID 142) −mir-186 (SEQ ID 112) − mir-187 (SEQ ID 143) − mir-190a (SEQ ID 144) −mir-191 (SEQ ID 145) − mir-301a (SEQ ID 146) − mir-325 (SEQ ID 147) −mir-331 (SEQ ID 148) − mir-345 (SEQ ID 149) − mir-361 (SEQ ID 150) −mir-374b (SEQ ID 80) − mir-380 (SEQ ID 151) − mir-381 (SEQ ID 152) −mir-450a-2 (SEQ ID 153) − mir-497a (SEQ ID 246) − mir-497-5p (SEQ ID241) − mir-505 (SEQ ID 156) − mir-551b (SEQ ID 157) − mir-664a-5p (SEQID 242) − mir-742 (SEQ ID 158) − mir-875 (SEQ ID 159) − mir-935 (SEQ ID160) − mir-17 (SEQ ID 107) + mir-21-5p (SEQ ID 243) + mir-23b (SEQ ID65) + mir-24-2 (SEQ ID 162) + mir-27b (SEQ ID 163) + mir-31 (SEQ ID164) + mir-34c (SEQ ID 165) + mir-129-1 (SEQ ID 166) + mir-140 (SEQ ID167) + mir-142-5p (SEQ ID 244) + mir-148a (SEQ ID 169) + mir-152 (SEQ ID170) + mir-184 (SEQ ID 171) + mir-199a-1 (SEQ ID 172) + mir-204 (SEQ ID173) + mir-212 (SEQ ID 174) + mir-214 (SEQ ID 175) + mir-375 (SEQ ID176) + mir-455 (SEQ ID 177) + mir-711 (SEQ ID 178) + mir-882 (SEQ ID179) +

In a further preferred embodiment, said method is a method for diagnosisof glioblastoma in an individual, wherein a pattern of at least 33,preferably of at least 44 of the miRNA listed in Table 11 is anindicator of glioblastoma, preferably said pattern is the pattern listedin Table 11.

TABLE 11 glioblastoma, MVs pattern (+, upregulated; −, downregulated)miRNA mir-29b-1 (SEQ ID 42) − mir-32 (SEQ ID 207) − mir-34a (SEQ ID 94)− mir-100 (SEQ ID 44) − mir-124-1 (SEQ ID 138) − mir-125a (SEQ ID 208) −mir-128-1 (SEQ ID 122) − mir-128-2 (SEQ ID 209) − mir-129-1 (SEQ ID 166)− mir-132 (SEQ ID 66) − mir-135a-1 (SEQ ID 210) − mir-137 (SEQ ID 99) −mir-138-1 (SEQ ID 123) − mir-139 (SEQ ID 211) − mir-146b (SEQ ID 54) −mir-149 (SEQ ID 212) − mir-181a-2 (SEQ ID 213) − mir-181b-1 (SEQ ID 140)− mir-181d (SEQ ID 141) − mir-184 (SEQ ID 171) − mir-185 (SEQ ID 142) −mir-218-1 (SEQ ID 124) − mir-326 (SEQ ID 214) − mir-483 (SEQ ID 215) −mir-491 (SEQ ID 216) − let-7c (SEQ ID 217) + mir-9-1 (SEQ ID 218) +mir-15b (SEQ ID 219) + mir-16-1 (SEQ ID 220) + mir-17 (SEQ ID 107) +mir-19b-1 (SEQ ID 71) + mir-20a (SEQ ID 108) + mir-21 (SEQ ID 221) +mir-23a (SEQ ID 222) + mir-24-1 (SEQ ID 223) + mir-25 (SEQ ID 224) +mir-27a (SEQ ID 225) + mir-30b (SEQ ID 84) + mir-92a-1 (SEQ ID 226) +mir-93 (SEQ ID 227) + mir-103a-1 (SEQ ID 228) + mir-106b (SEQ ID 229) +mir-125b-1 (SEQ ID 230) + mir-146a (SEQ ID 88) + mir-150 (SEQ ID 231) +mir-155 (SEQ ID 100) + mir-182 (SEQ ID 77) + mir-183 (SEQ ID 69) +mir-210 (SEQ ID 232) + mir-221 (SEQ ID 117) + mir-223 (SEQ ID 78) +mir-328 (SEQ ID 201) + mir-381 (SEQ ID 152) + mir-451a (SEQ ID 105) +mir-718 (SEQ ID 233) + mir-335 (SEQ ID 234) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of meningitis in an individual, wherein a pattern of atleast 16, preferably of at least 21 of the miRNA listed in Table 12 isan indicator of meningitis, preferably said pattern is the patternlisted in Table 12.

TABLE 12 meningitis, microglial MVs pattern (+, upregulated; −,downregulated) miRNA mir-138-1 (SEQ ID 123) − mir-192 (SEQ ID 180) −mir-219a-1 (SEQ ID 181) − mir-383 (SEQ ID 182) − mir-466 (SEQ ID 236) −mir-542 (SEQ ID 183) − mir-700 (SEQ ID 184) − mir-705 (SEQ ID 185) −mir-762 (SEQ ID 186) − mir-1901 (SEQ ID 187) − mir-1928 (SEQ ID 188) −mir-3474 (SEQ ID 189) − let-7a-1 (SEQ ID 93) + mir-10b (SEQ ID 63) +mir-105 (SEQ ID 190) + mir-141 (SEQ ID 191) + mir-155 (SEQ ID 100) +mir-191 (SEQ ID 145) + mir-200c (SEQ ID 192) + mir-201 (SEQ ID 193) +mir-214 (SEQ ID 175) + mir-297b (SEQ ID 194) + mir-302c (SEQ ID 195) +mir-495 (SEQ ID 196) + mir-670 (SEQ ID 197) + mir-673 (SEQ ID 198) +mir-1934 (SEQ ID 199) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of traumatic brain injury in an individual, wherein apattern of at least 12 of the miRNA listed in Table 13 is an indicatorof traumatic brain injury, preferably said pattern is the pattern listedin Table 13.

TABLE 13 traumatic brain injury, microglial MVs pattern (+, upregulated;−, downregulated) miRNA mir-129-5p (SEQ ID 245) − mir-140 (SEQ ID 167) −mir-185 (SEQ ID 142) − mir-212 (SEQ ID 174) − mir-328 (SEQ ID 201) −mir-361 (SEQ ID 150) − mir-487b (SEQ ID 202) − let-7a-2 (SEQ ID 83) +let-7b (SEQ ID 203) + mir-19b-1 (SEQ ID 71) + mir-21-5p (SEQ ID 243) +mir-126 (SEQ ID 238) + mir-146a (SEQ ID 88) + mir-155 (SEQ ID 100) +mir-223 (SEQ ID 78) + mir-292b (SEQ ID 204) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of ALS in an individual, wherein a pattern of at least 4 ofthe miRNA listed in Table 14 is an indicator of ALS, preferably saidpattern is the pattern listed in Table 14.

TABLE 14 ALS, microglial MVs pattern (+, upregulated) miRNA mir-22 (SEQID 64) + mir-125b-2 (SEQ ID 205) + mir-146b (SEQ ID 54) + mir-155 (SEQID 100) + mir-214 (SEQ ID 175) + mir-365-1 (SEQ ID 206) +

In a further preferred embodiment, said method is a method for diagnosisor prognosis of depression in an individual, wherein a pattern of atleast 5 of the miRNA listed in Table 15 is an indicator of depression,preferably said pattern is the pattern listed in Table 15.

TABLE 15 depression, microglial MVs pattern (+, upregulated) miRNAmir-34a (SEQ ID 94) − mir-451a (SEQ ID 105) − mir-132 (SEQ ID 66) +mir-134 (SEQ ID 91) + mir-144 (SEQ ID 75) + mir-182 (SEQ ID 77) +mir-221 (SEQ ID 117) +

The expression levels of a plurality of miRNAs are determined asexpression level values and, in a further preferred embodiment, saidstep d) comprises mathematically combining the expression level valuesof said plurality miRNAs by applying an algorithm to obtain a normalizedexpression level relative to at least one reference pattern ofexpression levels.

In a preferred embodiment, the determination of the expression profilein said step c) is obtained by the use of a method selected from thegroup consisting of a Sequencing-based method, an array based method anda PCR based method.

In a further aspect, a kit for diagnosis and prognosis ofneurodegenerative, neurological and inflammation-based diseases isdescribed, comprising:

a) means for determining the miRNA expression profile of a miRNA sampleof microglial microvesicles of a subject, and

b) at least one reference set of miRNA profile characteristic for aparticular condition.

EXAMPLES

Materials and Methods

Microglial Cell Cultures

Microglial cells were obtained from mixed glial cultures of P2 postnatalCD1 mice (Harlan Laboratories). Cortices were isolated in ice-coldbalanced salt solution (HBSS) without Ca⁺⁺ and Mg⁺⁺. Brains werecollected and meninges were manually removed under dissectingmicroscopes under sterile hood. The tissues were then finely choppedusing a scalpel. All of these operations were carried out at 4° C.

The tissue fragments were incubated at 37° C. for 20-30 min in a HBSSsolution with 2.5 mg/ml trypsin, 0.2 mg/ml EDTA, 1 mg/ml glucose and 0.1mg/ml bovine pancreatic DNase I in persistent gentle shaking in a waterbath incubator.

Following incubation, fresh culture medium (DMEM/F12 (3:1) containing20% heat-inactivated fetal bovine serum, 100 U/mlpenicillin-streptomycin was added, and the suspension was centrifuged at1500 rpm for 5 min at 4° C. Finally, single cell suspension was obtainedby manual resuspension of the pellet using a sterile Pasteur pipette.

The dissociated cells were plated onto poly-L-lysine coated flaskssupplemented with 20% heat-inactivated fetal bovine serum and 100 U/mlpenicillin, 10 mg/ml streptomycin, and 5.5 g/L glucose (glial medium).Purified microglial cultures were harvested by shaking 3-week-old mixedglial cultures. Detached microglia was seeded on poly-L-lysine-coatedflasks.

Purity of microglial cultures was carried out by immunocytochemicalanalysis of specific cellular markers: Antibodies against glialfibrillary acidic protein (GFAP) (1:400) for astrocytes, IB4 (1:100) formicroglia, olig2 for oligodendrocytes.

Abeta Amyloid Preparation

Beta Amyloid 1-42 (American Peptide) is prepared as recommended in thedata sheet and in literature; briefly the lyophilized peptide isdissolved in HPLC grade water at 6 mg/mL and then diluted to 1 mg/mLwith PBS 1× without Calcium and Magnesium. After 48 h of incubation (37°C.) the peptide is incubated with the cells at a final concentration of5 μM.

Isolation of CSF MVs

Approximately 4,500,000 cells/condition were primed with the differentexperimental stimuli (5 μM of Abeta oligomers for 24 hours to model AD,1 hour of 20 μM 6-OHDA to model PD, exposure for 2 hours to oxygenglucose deprivation protocol to model ischemia, exposure to GABA-Aantagonist such as 100 μM bicuculline for 2 hours to model Tourette'sSyndrome, challenge the primary microglia in microfluidic connectionwith dorsal root ganglion cells and challenged with 1 mM ATP to modelneuropathic pain, exposure to 10 nM IL6 for 2 hours to model autism,exposure to 30 ng/ml IL1beta and 100 nM TNFalfa for 2 hours to modelmultiple sclerosis, exposure for 2 hours to 100 μM BzATP of primarymicroglia cells processed with Mecp2 siRNA to model Rett Syndrome,exposure to 1 mM 3-nitropropionic acid for 24 hours to model HD,exposure to 500 μM kainic acid for 2 hours to model epilepsy, exposureto 100 ng/ml LPS for 24 hours to model meningitis, exposure of primarymicroglia cells to oxygen glucose deprivation protocol for 2 hoursfollowed by reperfusion in normoxic conditions to model traumatic braininjury, challenge for 30 minutes with 100 μM BzATP of primary microgliacells processed with SOD siRNA to model ALS, challenging of primaryhippocampal microglia cells processed with 100 ng/ml LPS for 4 hours and1 mM ATP for 30 minutes to model depression. Control cells (Untreated)were kept in culture media without triggering stimuli. Followingpriming, cells were triggered with 100 μM BzATP for 30 minutes undergentle rotation in KRH containing solution. The supernatant, containingshed vesicles, was withdrawn and incubated for 10 min at 4° C. undergentle periodic rotation with streptavidin beads, pre-coated withbiotinylated Annexin V. Shed vesicles bound to Annexin-coated beads werethen separated from the supernatant by gravity sedimentation at 4° C.

RNA Extraction from Isolated MVs

Total RNA (triplicate of biological samples) were extracted fromisolated MVs collected in a RNA preserving solution using Trizol LSReagent. Total RNA samples were analyzed by capillary electrophoresis onRNA 6000 Nano chip on Agilent Bioanalyzer 2100 Instrument and theirintegrity was checked through calculation of RNA Integrity Number (RIN).The qualitative control of RNAs obtained from MVs was performed using aRNA 6000 Pico chip on Bioanalyzer 2100 Instrument.

Library Preparation and Sequencing

The sequencing activities were characterized by two main steps: librarypreparation and their sequencing. The libraries were prepared usingTruSeq Small RNA Sample Preparation, a dedicated kit by Illumina,starting from total RNA as input material. The protocol takes advantageof the natural structure common to most known miRNA molecules andselectively enriches specifically in miRNAs. Each library was added witha unique index sequence and checked on Bioanalyzer 2100. Pooledlibraries were sequenced on Illumina platform in single read protocolwith the production of sequences of 36 bp in length.

Data Analysis

Small RNA sequencing data were processed from raw FASTQ files. UsingFastX toolkit, adaptors sequences were clipped from 3′ end of each read.Reads with low complexity and with a length less than 16 nucleotideswere discarded. Reads passing this QC step were aligned to the hg19human genome build allowing no mismatches in the seed region anddiscarding reads with more than 10 multi-mapping hits. Remnants readswere annotated and counted based on genomic annotations. Readsrealigning to miRBase were used for differential expression analysis.Normalizations and differential expression tests were performed withBioconductor Packages.

Results

MicroRNA content of MVs isolated from microglial cells challenged withdifferent neurodegenerative scenarios has been analysed.

Microglial cells obtained from rodent model have been primed to selectedinflammatory scenarios. MVs release has then be stimulated with 100 μMBzATP.

Isolated MVs were processed, RNA was isolated and microRNA analysis wascarried out. Specific miRNA patterns were evaluated.

Raw data was background-subtracted, Log 2-transformed, and normalized onread per million base. Values for each pathological indication werecompared to control, i.e. untreated cells, which were not primed butonly exposed to 100 μM BzATP triggering.

MiRNA with ratio/control above 2 were considered upregulated, whileMiRNA with ratio/control below 0.5 were considered downregulated.

Table 16 and 17 report the data observed in a AD microglial MVs model,i.e. Abeta-challenged microglial MVs with respect to control. In table16 are reported the Abeta-upregulated miRNA, in table 17 theAbeta-downregulated miRNA.

TABLE 16 RATIO RPM name Sequence (lib) AD/UT miR-125a-5pUCCCUGAGACCCUUUAACCUGUGA 1.51533566 (SEQ ID 1) miR-300-3pUAUGCAAGGGCAAGCUCUCUUC 1.521798302 (SEQ ID 2) miR-330-3pGCAAAGCACAGGGCCUGCAGAGA 1.530494292 (SEQ ID 3) miR-466n-3pUAUACAUGAGAGCAUACAUAGA 1.532714465 (SEQ ID 4) miR-501-5pAAUCCUUUGUCCCUGGGUGAAA 1.565278253 (SEQ ID 5) miR-146a-5pUGAGAACUGAAUUCCAUGGGUU 1.587610021 (SEQ ID 6) miR-24-1-5pGUGCCUACUGAGCUGAUAUCAGU 1.730044385 (SEQ ID 7) miR-1306-5pCACCACCUCCCCUGCAAACGUCC 1.81131115 (SEQ ID 8) miR-744-5pUGCGGGGCUAGGGCUAACAGCA 1.851357934 (SEQ ID 9) miR-671-5pAGGAAGCCCUGGAGGGGCUGGAG 2.030987686 (SEQ ID 10) miR-134-5pUGUGACUGGUUGACCAGAGGGG 2.06964569 (SEQ ID 11) miR-877-5pGUAGAGGAGAUGGCGCAGGG 2.173997574 (SEQ ID 12)

TABLE 17 RATIO RPM name Sequence (lib) AD/UT miR-23b-5pGGGUUCCUGGCAUGCUGAUUU 0.368062845 (SEQ ID 13) miR-669c-5pAUAGUUGUGUGUGGAUGUGUGU 0.40871633 (SEQ ID 14) miR-29b-3pUAGCACCAUUUGAAAUCAGUGUU 0.425022958 (SEQ ID 15) miR-195a-5pUAGCAGCACAGAAAUAUUGGC 0.431994357 (SEQ ID 16) miR-151-5pUCGAGGAGCUCACAGUCUAGU 0.457756929 (SEQ ID 17) miR-374c-3pACUUAGCAGGUUGUAUUAU 0.480963414 (SEQ ID 18) miR-6539GCACAGUGAUGAACUCUGAGGGCU 0.493012342 (SEQ ID 19)

Table 18 and 19 report the data observed in a PD microglial MVs model,i.e. 6-OHDA-challenged microglial MVs with respect to control. In table18 are reported the 6-OHDA-upregulated miRNA, in table 19 the6-OHDA-downregulated miRNA.

TABLE 18 RATIO RPM name Sequence (lib) AD/UT miR-16-1-3pCCAGUAUUGACUGUGCUGCUGA 2.014952955 (SEQ ID 20) miR-6399UUGCAAUGAUGGUAUUCUGAGG 2.218515684 (SEQ ID 21) miR-6240CCAAAGCAUCGCGAAGGCCCACGGCG 2.484737566 (SEQ ID 22) miR-23a-5pGGGGUUCCUGGGGAUGGGAUUU 2.588268298 (SEQ ID 23) miR-92a-1-5pAGGUUGGGAUUUGUCGCAAUGCU 2.711519169 (SEQ ID 24) miR-219a-1-3pAGAGUUGCGUCUGGACGUCCCG 2.803957322 (SEQ ID 25) miR-128-1-5pCGGGGCCGUAGCACUGUCUGA 3.822673178 (SEQ ID 26)

TABLE 19 RATIO RPM name Sequence (lib) AD/UT miR-1949CUAUACCAGGAUGUCAGCAUAGUU 0.246501743 (SEQ ID 27) miR-29b-3pUAGCACCAUUUGAAAUCAGUGUU 0.431888556 (SEQ ID 15) miR-872-3pUGAACUAUUGCAGUAGCCUCCU 0.454525164 (SEQ ID 28) miR-582-3pUAACCUGUUGAACAACUGAAC 0.456753229 (SEQ ID 29) miR-338-5pAACAAUAUCCUGGUGCUGAGUG 0.464103281 (SEQ ID 30) miR-379-5pUGGUAGACUAUGGAACGUAGG 0.477834147 (SEQ ID 31) miR-155-5pUUAAUGCUAAUUGUGAUAGGGGU 0.482096718 (SEQ ID 32) miR-450a-5pUUUUGCGAUGUGUUCCUAAUAU 0.482677061 (SEQ ID 33) miR-100-5pAACCCGUAGAUCCGAACUUGUG 0.485300306 (SEQ ID 34) miR-152-3pUCAGUGCAUGACAGAACUUGG 0.493828596 (SEQ ID 35) miR-222-3pAGCUACAUCUGGCUACUGGGU 0.498981358 (SEQ ID 36)

Table 20 and 21 report the data observed in an ischemic microglial MVsmodel, i.e. oxygen glucose deprived microglial MVs with respect tocontrol. The cells have been exposed to the oxygen glucose deprivationprotocol according to Kichev et al. J Biol Chem. 2014 289(13):9430-9439. In table 20 are reported the oxygen glucosedeprived-upregulated miRNA, in table 21 the oxygen glucosedeprived-downregulated miRNA.

TABLE 20 RATIO RPM name Sequence (lib) AD/UT mir-146bUGAGAACUGAAUUCCAUAGGCU 1.62 (SEQ ID 54) mir-188 CAUCCCUUGCAUGGUGGAGGG1.59 (SEQ ID 55) mir-346 UGUCUGCCCGAGUGCCUGCCUCU 2.1 (SEQ ID 56)mir-466f-3 UACGUGUGUGUGCAUGUGCAUG 1.6 (SEQ ID 57) mir-541AAGGGAUUCUGAUGUUGGUCACACU 1.49 (SEQ ID 58) mir-706AGAGAAACCCUGUCUCAAAAAA 3.45 (SEQ ID 59) mir-712 CUCCUUCACCCGGGCGGUACC2.45 (SEQ ID 60) mir-714 CGACGAGGGCCGGUCGGUCGC 2.36 (SEQ ID 61) mir-1224GUGAGGACUGGGGAGGUGGAG 1.82 (SEQ ID 62)

TABLE 21 RATIO RPM name Sequence (lib) AD/UT let-7eUGAGGUAGGAGGUUGUAUAGUU 0.57 (SEQ ID 37) mir-18b UAAGGUGCAUCUAGUGCUGUUAG0.59 (SEQ ID 38) mir-19a UAGUUUUGCAUAGUUGCACUAC 0.51 (SEQ ID 39) mir-21bUAGUUUAUCAGACUGAUAUUUCC 0.65 (SEQ ID 40) mir-26b UUCAAGUAAUUCAGGAUAGGU0.60 (SEQ ID 41) mir-29b-1 GCUGGUUUCAUAUGGUGGUUUA 0.64 (SEQ ID 42)mir-30c-1 UGUAAACAUCCUACACUCUCAGC 0.57 (SEQ ID 43) mir-100AACCCGUAGAUCCGAACUUGUG 0.6 (SEQ ID 44) mir-130a GCUCUUUUCACAUUGUGCUACU0.63 (SEQ ID 45) mir-181c AACAUUCAACCUGUCGGUGAGU 0.25 (SEQ ID 46)mir-297a-1 AUGUAUGUGUGCAUGUGCAUGU 0.46 (SEQ ID 47) mir-330UCUCUGGGCCUGUGUCUUAGGC 0.61 (SEQ ID 48) mir-342 AGGGGUGCUAUCUGUGAUUGAG0.55 (SEQ ID 49) mir-484 UCAGGCUCAGUCCCCUCCCGAU 0.5 (SEQ ID 50) mir-669bAGUUUUGUGUGCAUGUGCAUGU 0.31 (SEQ ID 51) mir-669e UGUCUUGUGUGUGCAUGUUCAU0.41 (SEQ ID 52) mir-708 CAACUAGACUGUGAGCUUCUAG 0.68 (SEQ ID 53)

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman. The only exceptions are miR-21b whose human homologue is miR-21UAGCUUAUCAGACUGAUGUUGA (SEQ ID 221), mir-297a-1 whose human homologue ismir-297 AUGUAUGUGUGCAUGUGCAUG (SEQ ID 235) and mir-466f-3, whose humanhomologue is miR-466 AUACACAUACACGCAACACACAU (SEQ ID 236).

Table 22 and 23 report the data observed in a Tourette's Syndromemicroglial MVs model, i.e. bicuculline exposed microglial MVs withrespect to control. Cells have been exposed to 100 μM bicuculline for 2hours, according to the protocol described in Frick et al. Brain BehavImmun. 2016 57:326-37. In table 22 are reported thebicuculline-upregulated miRNA, in table 23 the bicuculline-downregulatedmiRNA.

TABLE 22 RATIO RPM name Sequence (lib) AD/UT mir-19b-1AGUUUUGCAGGUUUGCAUCCAGC 1.86 (SEQ ID 71) mir-30a UGUAAACAUCCUCGACUGGAAG5 (SEQ ID 72) mir-33 GUGCAUUGUAGUUGCAUUGCA 1.35 (SEQ ID 73) mir-99bCACCCGUAGAACCGACCUUGCG 2.38 (SEQ ID 74) mir-144 GGAUAUCAUCAUAUACUGUAAGU1.48 (SEQ ID 75) mir-151 CUAGACUGAGGCUCCUUGAGG 1.21 (SEQ ID 76) mir-182UUUGGCAAUGGUAGAACUCACACCG 1.47 (SEQ ID 77) mir-223CGUGUAUUUGACAAGCUGAGUUG 1.60 (SEQ ID 78) mir-340 UCCGUCUCAGUUACUUUAUAGC2.09 (SEQ ID 79) mir-374b AUAUAAUACAACCUGCUAAGUG 2.3 (SEQ ID 80)mir-1247 CGGGAACGUCGAGACUGGAGC 2.41 (SEQ ID 82) mir-432UCUUGGAGUAGAUCAGUGGGCAG 1.37 (SEQ ID 81)

TABLE 23 RATIO RPM name Sequence (lib) AD/UT mir-10bUACCCUGUAGAACCGAAUUUGUG 0.45 (SEQ ID 63) mir-22 AGUUCUUCAGUGGCAAGCUUUA0.29 (SEQ ID 64) mir-23b GGGUUCCUGGCAUGCUGAUUU 0.19 (SEQ ID 65) mir-132AACCGUGGCUUUCGAUUGUUAC 0.38 (SEQ ID 66) mir-148bGAAGUUCUGUUAUACACUCAGGCU 0.13 (SEQ ID 67) mir-154 UAGGUUAUCCGUGUUGCCUUCG0.37 (SEQ ID 68) mir-183 UAUGGCACUGGUAGAAUUCACU 0.26 (SEQ ID 69) mir-337CGGCGUCAUGCAGGAGUUGAUU 0.50 (SEQ ID 70) mir-1224 GUGAGGACUGGGGAGGUGGAG0.38 (SEQ ID 62)

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman. The only exception is miR-151 whose human homologue is miR-151aCUAGACUGAAGCUCCUUGAGG (SEQ ID 237).

Table 24 and 25 report the data observed in a neuropathic painmicroglial MVs model, i.e. on microglial MVs obtained from primarymicroglia in microfluidic connection with dorsal root ganglion cells andchallenged with ATP with respect to control. Cells have been challengedwith 1 mM ATP for 30 min. according to Yamashita et al. PLoS One. 201611:10. In table 24 are reported the challenge-upregulated miRNA, intable 25 the challenge-downregulated miRNA.

TABLE 24 RATIO RPM (lib) name Sequence AD/UT mir-126b (SEQ ID 90)AUUAUUACUCACGGUACGAGUU 1.75 mir-134 (SEQ ID 91) UGUGACUGGUUGACCAGAGGGG1.64 mir-320 (SEQ ID 92) GCCUUCUCUUCCCGGUUCUUCC 2.24 mir-374b (SEQ ID80) AUAUAAUACAACCUGCUAAGUG 2.10

TABLE 25 RATIO RPM name Sequence (lib) AD/UT let-7a-2UGAGGUAGUAGGUUGUAUAGUU 0.45 (SEQ ID 83) mir-30b UGUAAACAUCCUACACUCAGCU0.40 (SEQ ID 84) mir-103-2 AGCUUCUUUACAGUGCUGCCUUG 0.53 (SEQ ID 85)mir-107 AGCUUCUUUACAGUGUUGCCUUG 0.359 (SEQ ID 86) mir-142aCAUAAAGUAGAAAGCACUACU 0.162 (SEQ ID 87) mir-146a UGAGAACUGAAUUCCAUGGGUU0.35 (SEQ ID 88) mir-151 UCGAGGAGCUCACAGUCUAGU 0.102 (SEQ ID 76)mir-374c AUAAUACAACCUGCUAAGUG 0.193 (SEQ ID 89)

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman. The only exceptions are miR-126b whose human homologue is miR-126CAUUAUUACUUUUGGUACGCG (SEQ ID 238) and miR-320 whose human homologue ismiR-320a AAAAGCUGGGUUGAGAGGGCGA (SEQ ID 239). Table 26 and 27 report thedata observed in an autism microglial MVs model, i.e. on IL6 exposedmicroglial MVs with respect to control. Cells have been exposed to 10 nMIL6 for 2 hours according to Tsilioni et al. Transl Psychiatry. 2015; 5.In table 26 are reported the IL6-upregulated miRNA, in table 27 theIL6-downregulated miRNA.

TABLE 26 RATIO RPM name Sequence (lib) AD/UT let-7f-1UGAGGUAGUAGAUUGUAUAGUU 2.43 (SEQ ID 97) mir-19a UAGUUUUGCAUAGUUGCACUAC3.54 (SEQ ID 39) mir-19b-2 AGUUUUGCAGAUUUGCAGUUCAGC 2.19 (SEQ ID 98)mir-21b UAGUUUAUCAGACUGAUAUUUCC 4.20 (SEQ ID 40) mir-22AGUUCUUCAGUGGCAAGCUUUA 1.76 (SEQ ID 64) mir-137 ACGGGUAUUCUUGGGUGGAUAAU3.66 (SEQ ID 99) mir-142a CAUAAAGUAGAAAGCACUACU 1.67 (SEQ ID 87) mir-144GGAUAUCAUCAUAUACUGUAAGU 5.43 (SEQ ID 75) mir-146b UGAGAACUGAAUUCCAUAGGCU3.26 (SEQ ID 154) mir-155 UUAAUGCUAAUUGUGAUAGGGGU 3.92 (SEQ ID 100)mir-219b AGAUGUCCAGCCACAAUUCUCG 1.22 (SEQ ID 101) mir-338AACAAUAUCCUGGUGCUGAGUG 3.88 (SEQ ID 102) mir-376c GUGGAUAUUCCUUCUAUGUUUA3.58 (SEQ ID 103) mir-379 UGGUAGACUAUGGAACGUAGG 6.86 (SEQ ID 104)mir-451a AAACCGUUACCAUUACUGAGUU 3.57 (SEQ ID 105) mir-494AGGUUGUCCGUGUUGUCUUCUC 3.45 (SEQ ID 106)

TABLE 27 RATIO RPM name Sequence (lib) AD/UT let-7a-1UGAGGUAGUAGGUUGUAUAGUU 0.54 (SEQ ID 93) mir-34a UGGCAGUGUCUUAGCUGGUUGU0.55 (SEQ ID 94) mir-92b AGGGACGGGACGUGGUGCAGUGUU 0.7 (SEQ ID 95)mir-211 UUCCCUUUGUCAUCCUUUGCCU 0.34 (SEQ ID 96)

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman. The only exception is miR-21b whose human homologue is miR-21UAGCUUAUCAGACUGAUGUUGA (SEQ ID 221).

Table 28 and 29 report the data observed in a MS microglial MVs model,i.e. on IL1beta and TNFalpha exposed microglial MVs with respect tocontrol. Cells have been exposed to 30 ng/mL IL1beta and 100 nM TNFalphafor 2 hours according to Xie et al. Glia, 2016 64:4, 583-602. In table28 are reported the IL1beta and TNFalpha-upregulated miRNA, in table 29the IL1beta and TNFalpha-downregulated miRNA.

TABLE 28 RATIO RPM name Sequence (lib) AD/UT let-7c-1UGAGGUAGUAGGUUGUAUGGUU 1.57 (SEQ ID 109) mir-20b CAAAGUGCUCAUAGUGCAGGUAG1.48 (SEQ ID 110) mir-142a UGUAGUGUUUCCUACUUUAUGGA 1.54 (SEQ ID 87)mir-145a AUUCCUGGAAAUACUGUUCUUG 3.1 (SEQ ID 111) mir-186GCCCUAAGGUGAAUUUUUUGGG 2.89 (SEQ ID 112) mir-223 UGUCAGUUUGUCAAAUACCCCA2.21 (SEQ ID 78) mir-664 UAUUCAUUUACUCCCCAGCCUA 5.25 (SEQ ID 113)

TABLE 29 RATIO RPM name Sequence (lib) AD/UT mir-17CAAAGUGCUUACAGUGCAGGUAG 0.35 (SEQ ID 107) mir-20aUAAAGUGCUUAUAGUGCAGGUAG 0.19 (SEQ ID 108)

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman.

Table 30 and 31 report the data observed in a Rett Syndrome microglialMVs model, i.e. on microglial MVs from primary microglia challenged withBzATP processed with Mecp2 siRNA with respect to control. The protocolis according to Lee Way et al. J. Neurosci. 2015, 35 (6) 2516-2529. Intable 30 are reported the challenge-upregulated miRNA, in table 31 thechallenge-downregulated miRNA.

TABLE 30 RATIO RPM name Sequence (lib) AD/UT mir-29b-1UAGCACCAUUUGAAAUCAGUGUU 3.12 (SEQ ID 42) mir-92bAGGGACGGGACGUGGUGCAGUGUU 2.16 (SEQ ID 95) mir-199bCCCAGUGUUUAGACUACCUGUUC 2.95 (SEQ ID 116) mir-221ACCUGGCAUACAAUGUAGAUUUCUGU 3.10 (SEQ ID 117) mir-296AGGGCCCCCCCUCAAUCCUGU 1.85 (SEQ ID 118) mir-329 AGAGGUUUUCUGGGUCUCUGUU2.96 (SEQ ID 119) mir-382 GAAGUUGUUCGUGGUGGAUUCG 3.51 (SEQ ID 120)

TABLE 31 RATIO RPM (lib) name Sequence AD/UT mir-122 (SEQ ID 114)AAACGCCAUUAUCACACUAA 0.45 mir-130a (SEQ ID 45) GCUCUUUUCACAUUGUGCUACU0.21 mir-146a (SEQ ID 88) UGAGAACUGAAUUCCAUGGGUU 0.22 mir-146b (SEQ ID54) UGAGAACUGAAUUCCAUAGGCU 0.42 mir-342 (SEQ ID 49)AGGGGUGCUAUCUGUGAUUGAG 0.42 mir-409 (SEQ ID 115) AGGUUACCCGAGCAACUUUGC0.25 AU

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman. The only exception is miR-329 whose human homologue is miR-329-5pGAGGUUUUCUGGGUUUCUGUUUC (SEQ ID 240).

Table 32 and 33 report the data observed in a HD microglial MVs model,i.e. on 3NP exposed microglial MVs with respect to control. Cells havebeen exposed to 1 mM 3-nitropropionic acid for 24 hours according to Ruyet al. Neurobiol. Dis. 2003:12 121-132. In table 32 are reported the3NP-upregulated miRNA, in table 33 the 3NP-downregulated miRNA.

TABLE 32 RATIO RPM name Sequence (lib) AD/UT mir-34aUGGCAGUGUCUUAGCUGGUUGU 1.73 (SEQ ID 94) mir-207 GCUUCUCCUGGCUCUCCUCCCUC1.72 (SEQ ID 129) mir-18a UAAGGUGCAUCUAGUGCAGAUAG 1.97 (SEQ ID 130)mir-448 UUGCAUAUGUAGGAUGUCCCAU 2.11 (SEQ ID 131) mir-669cUACACACACACACACAAGUAAA 2.16 (SEQ ID 133)

TABLE 33 RATIO RPM name Sequence (lib) AD/UT mir-22 (SEQ ID 64)AGUUCUUCAGUGGCAAGCUUUA 1.27 mir-29c (SEQ ID 121) UGACCGAUUUCUCCUGGUGUUC0.97 mir-128-1 (SEQ ID 122) CGGGGCCGUAGCACUGUCUGA 0.76 mir-132 (SEQ ID66) AACCGUGGCUUUCGAUUGUUAC 0.93 mir-138-1 (SEQ ID 123)AGCUGGUGUUGUGAAUCAGGCCG 1.39 mir-218-1 (SEQ ID 124)UUGUGCUUGAUCUAACCAUGU 0.94 mir-222 (SEQ ID 125) UCAGUAGCCAGUGUAGAUCCU1.37 mir-344-1 (SEQ ID 126) UGAUCUAGCCAAAGCCUGACUGU 1.79 mir-466b-2 (SEQID 127) UGAUGUGUGUGUACAUGUACAU 0.39 mir-674 (SEQ ID 128)CACAGCUCCCAUCUCAGAACAA 0.51

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman. The only exception is miR-466b-2 whose human homologue is miR-466AUACACAUACACGCAACACACAU (SEQ ID 236).

Table 34 and 35 report the data observed in an epilepsy microglial MVsmodel, i.e. on kainic acid exposed microglial MVs with respect tocontrol. Cells have been exposed to 500 μM Kainic Acid for 2 hoursaccording to Zhang et al. Curr Neuropharmacol. 20119(2): 388-398. Intable 34 are reported the kainic acid-upregulated miRNA, in table 35 thekainic acid-downregulated miRNA.

TABLE 34 RATIO RPM name Sequence (lib) AD/UT mir-17 (SEQ ID 107)CAAAGUGCUUACAGUGCAGGUAG 2.87 mir-21a (SEQ ID 161) UAGCUUAUCAGACUGAUGUUGA3.56 mir-23b (SEQ ID 65) GGGUUCCUGGCAUGCUGAUUU 2.98 mir-24-2 (SEQ ID162) GUGCCUACUGAGCUGAAACAGU 2.05 mir-27b (SEQ ID 163)AGAGCUUAGCUGAUUGGUGAAC 3.51 mir-31 (SEQ ID 164) AGGCAAGAUGCUGGCAUAGCUG4.21 mir-34c (SEQ ID 165) AGGCAGUGUAGUUAGCUGAUUGC 2.16 mir-129-1 (SEQ ID166) CUUUUUGCGGUCUGGGCUUGC 1.87 mir-140 (SEQ ID 167)CAGUGGUUUUACCCUAUGGUAG 2.25 mir-142b (SEQ ID 168) UCCAUAAAGUAGGAAACACU2.64 mir-148a (SEQ ID 169) AAAGUUCUGAGACACUCCGACU 3.66 mir-152 (SEQ ID170) UAGGUUCUGUGAUACACUCCGACU 3.11 mir-184 (SEQ ID 171)CCUUAUCACUUUUCCAGCCAGC 3.13 mir-199a-1 (SEQ ID 172)CCCAGUGUUCAGACUACCUGUUC 3.22 mir-204 (SEQ ID 173) UUCCCUUUGUCAUCCUAUGCCU2.73 mir-212 (SEQ ID 174) ACCUUGGCUCUAGACUGCUUACU 3.14 mir-214 (SEQ ID175) UGCCUGUCUACACUUGCUGUGC 1.23 mir-375 (SEQ ID 176)GCGACGAGCCCCUCGCACAAAC 2.05 mir-455 (SEQ ID 177) UAUGUGCCUUUGGACUACAUCG2.16 mir-711 (SEQ ID 178) GGGACCCGGGGAGAGAUGUAAG 2.68 mir-882 (SEQ ID179) AGGAGAGAGUUAGCGCAUUAGU 1.62

TABLE 35 RATIO RPM name Sequence (lib) AD/UT let-7d (SEQ ID 134)AGAGGUAGUAGGUUGCAUAGUU 0.25 let-7f-1 (SEQ ID 97) UGAGGUAGUAGAUUGUAUAGUU0.34 mir-30a (SEQ ID 72) UGUAAACAUCCUCGACUGGAAG 0.23 mir-30e (SEQ ID135) UGUAAACAUCCUUGACUGGAAG 0.2 mir-34b (SEQ ID 136)AGGCAGUGUAAUUAGCUGAUUGU 0.15 mir-98 (SEQ ID 137) CUAUACAACUUACUACUUUCCU0.49 mir-124-1 (SEQ ID 138) CGUGUUCACAGCGGACCUUGAU 0.28 mir-181a-1 (SEQID 139) AACAUUCAACGCUGUCGGUGAGU 0.73 mir-181b-1 (SEQ ID 140)AACAUUCAUUGCUGUCGGUGGGU 0.88 mir-181d (SEQ ID 141)AACAUUCAUUGUUGUCGGUGGGU 0.41 mir-185 (SEQ ID 142) UGGAGAGAAAGGCAGUUCCUGA0.22 mir-186 (SEQ ID 112) CAAAGAAUUCUCCUUUUGGGCU 0.46 mir-187 (SEQ ID143) AGGCUACAACACAGGACCCGGG 0.18 mir-190a (SEQ ID 144)UGAUAUGUUUGAUAUAUUAGGU 0.33 mir-191 (SEQ ID 145) CAACGGAAUCCCAAAAGCAGCUG0.51 mir-301a (SEQ ID 146) GCUCUGACUUUAUUGCACUACU 0.33 mir-325 (SEQ ID147) CCUAGUAGGUGCUCAGUAAGUGU 0.25 mir-331 (SEQ ID 148)CUAGGUAUGGUCCCAGGGAUCC 0.41 mir-345 (SEQ ID 149) GCUGACCCCUAGUCCAGUGCUU0.31 mir-361 (SEQ ID 150) UUAUCAGAAUCUCCAGGGGUAC 0.24 mir-374b (SEQ ID80) AUAUAAUACAACCUGCUAAGUG 0.20 mir-380 (SEQ ID 151)AUGGUUGACCAUAGAACAUGCG 0.31 mir-381 (SEQ ID 152) AGCGAGGUUGCCCUUUGUAUAUU0.27 mir-450a-2 (SEQ ID 153) UUUUGCGAUGUGUUCCUAAUAU 0.41 mir-497a (SEQID 246) CAGCAGCACACUGUGGUUUGUA 0.27 mir-497b (SEQ ID 155)CACCACAGUGUGGUUUGGACGUGG 0.25 mir-505 (SEQ ID 156)GGGAGCCAGGAAGUAUUGAUGUU 0.37 mir-551b (SEQ ID 157)GAAAUCAAGCUUGGGUGAGACCU 0.32 mir-664 (SEQ ID 113) CUGGCUGGGGAAAAUGACUGG0.53 mir-742 (SEQ ID 158) UACUCACAUGGUUGCUAAUCA 0.36 mir-875 (SEQ ID159) UAUACCUCAGUUUUAUCAGGUG 0.23 mir-935 (SEQ ID 160)CCCAGUUACCGCUUCCGCUACCGC 0.47

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman. The only exceptions are miR-497b whose human homologue ismiR-497-5p CAGCAGCACACUGUGGUUUGU (SEQ ID 241), miR-664 whose humanhomologue is miR-664a-5p ACUGGCUAGGGAAAAUGAUUGGAU (SEQ ID 242), miR-21a,human homologue miR-21-5p UAGCUUAUCAGACUGAUGUUGA (SEQ ID 243) andmiR-142b whose human homologue is miR-142-5p CAUAAAGUAGAAAGCACUACU (SEQID 244).

Table 36 and 37 report the data observed in a meningitis microglial MVsmodel, i.e. on LPS exposed microglial MVs with respect to control. Cellshave been exposed to 100 ng/ml LPS for hours according to Bianco F. etal. J. Immunol. 2005 174, 11:7268-7277. In table 36 are reported theLPS-upregulated miRNA, in table 37 the LPS-downregulated miRNA.

TABLE 36 RATIO RPM name Sequence (lib) AD/UT mir-138-1 (SEQ ID 123)AGCUGGUGUUGUGAAUCAGGCCG 0.33 mir-192 (SEQ ID 180) CUGACCUAUGAAUUGACAGCC0.31 mir-219a-1 (SEQ ID 181) UGAUUGUCCAAACGCAAUUCU 0.21 mir-383 (SEQ ID182) AGAUCAGAAGGUGACUGUGGCU 0.35 mir-466b-3 (SEQ ID 132)UGAUGUGUGUGUACAUGUACAU 0.41 mir-542 (SEQ ID 183) CUCGGGGAUCAUCAUGUCACGA0.21 mir-700 (SEQ ID 184) UAAGGCUCCUUCCUGUGCUUGC 0.43 mir-705 (SEQ ID185) GGUGGGAGGUGGGGUGGGCA 0.31 mir-762 (SEQ ID 186)GGGGCUGGGGCCGGGACAGAGC 0.32 mir-1901 (SEQ ID 187) CCGCUCGUACUCCCGGGGGUCC0.15 mir-1928 (SEQ ID 188) AGCUACAUUGCCAGCUC 0.34 mir-3474 (SEQ ID 189)CCCUGGGAGGAGACGUGGAUUC 0.31

TABLE 37 RATIO RPM name Sequence (lib) AD/UT let-7a-1UGAGGUAGUAGGUUGUAUAGUU 3.21 (SEQ ID 93) mir-10b UACCCUGUAGAACCGAAUUUGUG3.53 (SEQ ID 63) mir-105 CCAAGUGCUCAGAUGCUUGUGGU 2.43 (SEQ ID 190)mir-141 CAUCUUCCAGUGCAGUGUUGGA 2.45 (SEQ ID 191) mir-155UUAAUGCUAAUUGUGAUAGGGGU 3.15 (SEQ ID 100) mir-191CAACGGAAUCCCAAAAGCAGCUG 1.87 (SEQ ID 145) mir-200cCGUCUUACCCAGCAGUGUUUGG 2.86 (SEQ ID 192) mir-201 UACUCAGUAAGGCAUUGUUCUU3.16 (SEQ ID 193) mir-214 UGCCUGUCUACACUUGCUGUGC 1.73 (SEQ ID 175)mir-297b AUGUAUGUGUGCAUGAACAUGU 1.78 (SEQ ID 194) mir-302cGCUUUAACAUGGGGUUACCUGC 1.65 (SEQ ID 195) mir-495 GAAGUUGCCCAUGUUAUUUUUCG1.67 (SEQ ID 196) mir-670 AUCCCUGAGUGUAUGUGGUGAA 2.37 (SEQ ID 197)mir-673 CUCACAGCUCUGGUCCUUGGAG 3.24 (SEQ ID 198) mir-1934UCUGGUCCCCUGCUUCGUCCUCU 2.17 (SEQ ID 199)

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman. The only exception is miR-466b-3 whose human homologue is miR-466(SEQ ID 236).

Table 38 and 39 report the data observed in a Traumatic Brain injurymicroglial MVs model, i.e. on microglial MVs from microglia exposed tooxygen glucose deprivation protocol followed by reperfusion in normoxicconditions with respect to control. Primary microglia cells have beenexposed to 2 hours oxygen glucose deprivation protocol followed by 2hours of reperfusion in normoxic conditions, according to Kichev et al.J. Biol. Chem. 2014 289(13): 9430-9439. In table 38 are reported thechallenge-upregulated miRNA, in table 39 the challenge-downregulatedmiRNA.

TABLE 38 RATIO RPM name Sequence (lib) AD/UT let-7a-2UGAGGUAGUAGGUUGUAUAGUU 2.06 (SEQ ID 83) let-7b UGAGGUAGUAGGUUGUGUGGUU1.78 (SEQ ID 203) mir-19b-1 AGUUUUGCAGGUUUGCAUCCAGC 2.46 (SEQ ID 71)mir-21a UAGCUUAUCAGACUGAUGUUGA 2.35 (SEQ ID 161) mir-126bAUUAUUACUCACGGUACGAGUU 3.32 (SEQ ID 90) mir-146a UGAGAACUGAAUUCCAUGGGUU2.56 (SEQ ID 88) mir-155 UUAAUGCUAAUUGUGAUAGGGGU 2.54 (SEQ ID 100)mir-223 CGUGUAUUUGACAAGCUGAGUUG 3.21 (SEQ ID 78) mir-292bACUCAAAACCUGGCGGCACUUUU 4.32 (SEQ ID 204)

TABLE 39 RATIO RPM name Sequence (lib) AD/UT mir-129bCAGUGGUUUUACCCUAUGGUAG 0.43 (SEQ ID 200) mir-140 CAGUGGUUUUACCCUAUGGUAG0.22 (SEQ ID 167) mir-185 UGGAGAGAAAGGCAGUUCCUGA 0.27 (SEQ ID 142)mir-212 ACCUUGGCUCUAGACUGCUUACU 0.32 (SEQ ID 174) mir-328GGGGGGCAGGAGGGGCUCAGGG 0.23 (SEQ ID 201) mir-361 UUAUCAGAAUCUCCAGGGGUAC0.33 (SEQ ID 150) mir-487b UGGUUAUCCCUGUCCUCUUCG 0.26 (SEQ ID 202)

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman. The only exceptions are miR-129b whose human homologue ismiR-129-5p CUUUUUGCGGUCUGGGCUUGC (SEQ ID 245), miR-21a whose humanhomologue is miR-21-5p UAGCUUAUCAGACUGAUGUUGA (SEQ ID 243) and miR-126bwhose human homologue is miR-126 CAUUAUUACUUUUGGUACGCG (SEQ ID 238).

Table 40 reports the data observed in a ALS microglial MVs model, i.e.on microglial MVs from microglia challenged with BzATP and processedwith SOD siRNA with respect to control. Primary microglia cellsprocessed with SOD siRNA have been challenged with 100 μM BzATP for 30minutes, according to Brites et al. Front. Cell. Neurosci. 2014 8:117.In table 40 are reported the challenge-downregulated miRNA.

TABLE 40 RATIO RPM name Sequence (lib) AD/UT mir-22AGUUCUUCAGUGGCAAGCUUUA 2.15 (SEQ ID 64) mir-125b-2UCCCUGAGACCCUAACUUGUGA 2.47 (SEQ ID 205) mir-146b UGAGAACUGAAUUCCAUAGGCU3.21 (SEQ ID 54) mir-155 UUAAUGCUAAUUGUGAUAGGGGU 1.98 (SEQ ID 100)mir-214 UGCCUGUCUACACUUGCUGUGC 3.29 (SEQ ID 175) mir-365-1AGGGACUUUUGGGGGCAGAUGUG 2.08 (SEQ ID 206)

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman.

Tables 41 and 42 report the data observed in a depression microglial MVsmodel, i.e. on LPS and ATP exposed microglial MVs with respect tocontrol. Cells were processed with 100 mg/ml LPS and 1 mM ATP for 4hours, according to Brites et al. Front. Cell Neurosci. 2015 8:11. Intable 41 are reported the LPS and ATP-upregulated miRNA, in table 42 theLPS and ATP-downregulated miRNA.

TABLE 41 RATIO RPM name Sequence (lib) AD/UT mir-132AACCGUGGCUUUCGAUUGUUAC 2.16 (SEQ ID 66) mir-134 UGUGACUGGUUGACCAGAGGGG2.43 (SEQ ID 91) mir-144 GGAUAUCAUCAUAUACUGUAAGU 2.67 (SEQ ID 75)mir-182 UUUGGCAAUGGUAGAACUCACACCG 3.61 (SEQ ID 77) mir-221ACCUGGCAUACAAUGUAGAUUUCUGU 4.51 (SEQ ID 117)

TABLE 42 RATIO RPM name Sequence (lib) AD/UT mir-34aUGGCAGUGUCUUAGCUGGUUGU 0.15 (SEQ ID 94) mir-451a AAACCGUUACCAUUACUGAGUU0.43 (SEQ ID 105)

The above listed miRNA are mouse miRNA whose sequence is conserved inhuman.

Finally, Tables 43 and 44 report the data observed in MVs from U87glioblastoma human cell line, where glioblastoma cells are consideredmicroglial cells, with respect to standard microglia cells. In table 43are reported the upregulated miRNA, in table 44 the downregulated miRNA.

TABLE 43 RATIO RPM name Sequence (lib) AD/UT mir-29b-1 (SEQ ID 42)GCUGGUUUCAUAUGGUGGUUUAGA 0.4 mir-32 (SEQ ID 207) CAAUUUAGUGUGUGUGAUAUUU0.1 mir-34a (SEQ ID 94) UGGCAGUGUCUUAGCUGGUUGU 0.4 mir-100 (SEQ ID 44)AACCCGUAGAUCCGAACUUGUG 0.21 mir-124-1 (SEQ ID 138)CGUGUUCACAGCGGACCUUGAU 0.54 mir-125a (SEQ ID 208)UCCCUGAGACCCUUUAACCUGUGA 0.28 mir-128-1 (SEQ ID 122)CGGGGCCGUAGCACUGUCUGAGA 0.42 mir-128-2 (SEQ ID 209)GGGGGCCGAUACACUGUACGAGA 0.92 mir-129-1 (SEQ ID 166)CUUUUUGCGGUCUGGGCUUGC 0.45 mir-132 (SEQ ID 66) ACCGUGGCUUUCGAUUGUUACU0.17 mir-135a-1 (SEQ ID 210) UAUGGCUUUUUAUUCCUAUGUGA 0.37 mir-137 (SEQID 99) UUAUUGCUUAAGAAUACGCGUAG 0.31 mir-138-1 (SEQ ID 123)AGCUGGUGUUGUGAAUCAGGCCG 0.28 mir-139 (SEQ ID 211)UCUACAGUGCACGUGUCUCCAGU 0.15 mir-146b (SEQ ID 54) UGAGAACUGAAUUCCAUAGGCU0.41 mir-149 (SEQ ID 212) UCUGGCUCCGUGUCUUCACUCCC 0.56 mir-181a-2 (SEQID 213) AACAUUCAACGCUGUCGGUGAGU 0.21 mir-181b-1 (SEQ ID 140)AACAUUCAUUGCUGUCGGUGGGU 0.44 mir-181d (SEQ ID 141)AACAUUCAUUGUUGUCGGUGGGU 0.54 mir-184 (SEQ ID 171) UGGACGGAGAACUGAUAAGGGU0.16 mir-185 (SEQ ID 142) UGGAGAGAAAGGCAGUUCCUGA 0.35 mir-218-1 (SEQ ID124) UUGUGCUUGAUCUAACCAUGU 0.28 mir-326 (SEQ ID 214)CCUCUGGGCCCUUCCUCCAG 0.54 mir-483 (SEQ ID 215) AAGACGGGAGGAAAGAAGGGAG0.23 mir-491 (SEQ ID 216) AGUGGGGAACCCUUCCAUGAGG 0.43

TABLE 44 RATIO RPM name Sequence (lib) AD/UT let-7c (SEQ ID 217)UGAGGUAGUAGGUUGUAUGGUU 3.21 mir-9-1 (SEQ ID 218) UCUUUGGUUAUCUAGCUGUAUGA2.51 mir-15b (SEQ ID 219) UAGCAGCACAUCAUGGUUUACA 2.54 mir-16-1 (SEQ ID220) UAGCAGCACGUAAAUAUUGGCG 1.46 mir-17 (SEQ ID 107)CAAAGUGCUUACAGUGCAGGUAG 2.56 mir-19b-1 (SEQ ID 71)AGUUUUGCAGGUUUGCAUCCAGC 2.62 mir-20a (SEQ ID 108)UAAAGUGCUUAUAGUGCAGGUAG 2.68 mir-21 (SEQ ID 221) UAGCUUAUCAGACUGAUGUUGA2.07 mir-23a (SEQ ID 222) GGGGUUCCUGGGGAUGGGAUUU 1.86 mir-24-1 (SEQ ID223) UGCCUACUGAGCUGAUAUCAGU 3.21 mir-25 (SEQ ID 224)AGGCGGAGACUUGGGCAAUUG 2.58 mir-27a (SEQ ID 225) AGGGCUUAGCUGCUUGUGAGCA3.25 mir-30b (SEQ ID 84) UGUAAACAUCCUACACUCAGCU 1.56 mir-92a-1 (SEQ ID226) AGGUUGGGAUCGGUUGCAAUGCU 2.48 mir-93 (SEQ ID 227)CAAAGUGCUGUUCGUGCAGGUAG 2.67 mir-103a-1 (SEQ ID 228)AGCAGCAUUGUACAGGGCUAUGA 2.31 mir-106b (SEQ ID 229) UAAAGUGCUGACAGUGCAGAU2.65 mir-125b-1 (SEQ ID 230) UCCCUGAGACCCUAACUUGUGA 1.29 mir-146a (SEQID 88) UGAGAACUGAAUUCCAUGGGUU 2.19 mir-150 (SEQ ID 231)UCUCCCAACCCUUGUACCAGUG 1.38 mir-155 (SEQ ID 100) UUAAUGCUAAUCGUGAUAGGGGU1.53 mir-182 (SEQ ID 77) UUUGGCAAUGGUAGAACUCACACU 2.18 mir-183 (SEQ ID69) UAUGGCACUGGUAGAAUUCACU 2.07 mir-210 (SEQ ID 232)AGCCCCUGCCCACCGCACACUG 3.51 mir-221 (SEQ ID 117) ACCUGGCAUACAAUGUAGAUUU2.47 mir-223 (SEQ ID 78) CGUGUAUUUGACAAGCUGAGUU 3.15 mir-328 (SEQ ID201) GGGGGGGCAGGAGGGGCUCAGGG 2.51 mir-381 (SEQ ID 152)AGCGAGGUUGCCCUUUGUAUAU 4.21 mir-451a (SEQ ID 105) AAACCGUUACCAUUACUGAGUU2.31 mir-718 (SEQ ID 233) CUUCCGCCCCGCCGGGCGUCG 4.32 mir-335 (SEQ ID234) UCAAGAGCAAUAACGAAAAAUGU 5.24

The invention claimed is:
 1. A method for the in vitro diagnosis ofParkinson's Disease (PD) in an individual, wherein the method comprisesthe steps: a) isolating microglial microvesicles (MVs) from biologicalfluids obtained from an individual; b) collecting the miRNA contained insaid MVs; c) determining the expression profile of a predetermined setof miRNA, defining a microglial MVs miRNA pattern; d) comparing saidexpression profile to one or several reference expression profilesobtained from at least one healthy individual, or an individual nothaving Parkinson's Disease (PD); and, e) treating said individual with asuitable therapy based on the diagnosis of said individual; whereby adifference between said determined expression profile and said one orseveral reference expression profiles is indicative of Parkinson'sDisease (PD); whereby said microglial MVs miRNA pattern, with respect tosaid reference pattern, is characterised as follows: downregulatedmiRNA: miR-100-5p (SEQ ID 34), miR-152-3p (SEQ ID 35), miR-155-5p (SEQID 32), miR-1949 (SEQ ID 27), miR-501-5p (SEQ ID 5), miR-6399 (SEQ ID21), miR-872-3p (SEQ ID 28); upregulated miRNA: miR-128-1-5p (SEQ ID26), miR-16-1-3p (SEQ ID 20), miR-219a-1-3p (SEQ ID 25), miR-222-3p (SEQID 36), miR-23a-5p (SEQ ID 23), miR-338-5p (SEQ ID 30), miR-379-5p (SEQID 31), miR-450a-5p (SEQ ID 33), miR-582-3p (SEQ ID 29), miR-6240 (SEQID 22), miR-92a-1-5p (SEQ ID 24).
 2. The method according to claim 1,wherein said microglial MVs miRNA pattern, with respect to saidreference pattern, is characterised as follows: downregulated miRNA:miR-100-5p (SEQ ID 34), miR-152-3p (SEQ ID 35), miR-1949 (SEQ ID 27),miR-501-5p (SEQ ID 5); and, upregulated miRNA: miR-128-1-5p (SEQ ID 26),miR-16-1-3p (SEQ ID 20), miR-219a-1-3p (SEQ ID 25), miR-222-3p (SEQ ID36), miR-23a-5p (SEQ ID 23), miR-338-5p (SEQ ID 30), miR-379-5p (SEQ ID31), miR-450a-5p (SEQ ID 33), miR-582-3p (SEQ ID 29), miR-92a-1-5p (SEQID 24).